Method for analyzing enzyme-catalyzed reactions using MALDI-TOF mass spectrometry

ABSTRACT

A process is described for analyzing enzyme-catalyzed conversions of nonpolymeric substrates to nonpolymeric products with the aid of MALDI-TOF mass spectrometry, preferably in the presence of an internal standard on a specific carrier material.

This application is the US national phase of international applicationPCT/EP01/06416 filed 6 Jun. 2001 which designated the U.S.

The present invention relates to a process for analyzingenzyme-catalyzed conversions of nonpolymeric substrates to nonpolymericproducts with the aid of MALDI-TOF mass spectrometry, preferably in thepresence of an internal standard on a specific carrier material.

Success in the screening for novel enzymatic reactions depends to alarge extent on chance. This kind of screening demands the scrutinizingof a very large number of organisms for the desired enzymatic activityuntil the desired enzyme activity is found. Screening for these enzymeactivities therefore requires rapid, simple, highly sensitive and highlyspecific analytical processes.

A major problem in the screening for novel enzymatic activities is thequick and simple identification of the products generated in theenzymatic reaction and/or, where appropriate, the decrease in thesubstrate employed. Product analysis usually involves using separationprocesses such as thin layer chromatography (=TLC), high pressure liquidchromatography (=HPLC) or gas chromatography (=GC). Processes such asNMR which are usable after work-up via, for example, salt precipitationand/or subsequent chromatography may also be used for analysis. Theseprocesses are time-consuming and allow only a limited sample throughput,and therefore those analytical processes are not usable for so-calledhigh throughput screening (=HTS) which involves initial screening forthe desired reaction. Advantageously, these methods provide informationboth about the product and, where appropriate, about the decrease insubstrate.

In order to facilitate higher sample throughput in HTS, indirect,readily measurable processes such as color reactions in the visiblerange, turbidity measurements, fluorescence, conductivity measurementsetc. are frequently used. Although said processes are in principle verysensitive, they are also susceptible to faults. Particular disadvantageshere are the analysis of a large number of false positive samples inthis procedure and, since these are indirect detection processes, theabsence of any information about product and/or substrate. In order tobe able to exclude these false positives from the further procedure, itis common to use further analytical processes such as, for example, TLC,HPLC or GC after the first screening. This is again very time-consuming.

Generally it can be said that improving the sensitivity andmeaningfulness of detection processes regarding the reaction productsleads to the slowing down of an assay.

MALDI-TOF MS (=matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry) represents a quick and simple processwhich is used widely for analyzing large non-volatile biomolecules, inparticular such as peptides, proteins, oligonucleotides andoligosaccharides or other polymers. High molecular weight materials suchas tar, humic acid, fulvic acid or kerogens have also been analyzed byMALDI (Zenobi and Knochenmuss, Mass Spec. Rev., 1998, 17, 337-366).

Quantifying measurement results in MALDI MS is problematic, because theintensity of the signal depends to a high degree on the homogeneity ofthe applied sample and on the irradiation density of the laser (Ens etal., Rapid Commun. Mass Spectrom, 5, 1991: 117-123), the intensityincreasing at first approximation exponentially with increasing laserenergy (Ens et al., Rapid Commun. Mass Spectrom, 5, 1991: 117-123). Asignal intensity which is too high may possibly lead to signalsaturation at the detector, and this also rules out quantification.Besides these problems of physical and technical nature there are otherreasons which make a quantitative evaluation of MALDI measurementsdifficult. Thus, for example, fragments of the ions searched for ormolecule adducts may appear. The most serious problem in quantitativeMALDI MS, however, is the inhomogeneity of the samples. The quality of aMALDI spectrum is to a great extent dependent on the morphology of thesample studied (Garden & Sweedler, Anal. Chem. 72, 2000: 30-36). As aresult, it is possible to observe significant differences regarding theappearance of signals, the intensity, the resolution and the massaccuracy as soon as different sites of a MALDI sample are studied (Cohen& Chait, Anal. Chem., 68, 1996: 31-37; Strupat et al., Int. J. MassSpectrom. Ion Processes, 111, 1991: 89-102; Amado et al., Rapid Commun.Mass Spectrom., 11, 1997: 1347-1352). These inhomogeneities are based onan uneven distribution of matrix and analyte on the sample target, whichis caused by different crystallization behavior of these two components.In order to abolish or minimize these inhomogeneities—which istantamount to formation of a microcrystalline homogeneous sampletopology—a number of suggestions have been worked out previously. Theseinclude, for example, the use of comatrices (Gusev et al., Anal. Chem.,67, 1995: 1034-1041), multilayer preparations, the use of solventmixtures and electrospray preparations (Hensel et al., Rapid Commun.Mass Spectrom., 11, 1997: 1785-1793) (a compilation of these experimentscan be found in Garden & Sweedler, Anal. Chem. 72, 2000: 30-6). However,all of these approaches have limitations and are therefore not broadlyusable. Quantification therefore continues to be a problem.

In MALDI the samples are usually applied in a thin layer onto a metalsurface and then exposed to a pulsed laser. Focusing the emitted ionscan increase the resolution in the low mass region of the mass spectrato about 5000 Da.

Duncan et al. (Rapid Communications in Mass Spectrometry, Vol. 7, 1993:1090-1094) describe analyzing the low molecular weight polar compounds3,4-dihydroxyphenylalanine, acetylcholine and the peptideAc-Ser-Ile-Arg-His-Tyr-NH₂ with the aid of MALDI and in the presence ofinternal standards in the form of the corresponding ¹³C- and ²H-labeledcompounds and a similar peptide, respectively.

Goheen et al. (J. Mass Spec., Vol. 32, 1997: 820-828) describe the useof MALDI-TOF MS for analyzing the following low molecular weightcompounds:

Citric acid, propionic acid, butyric acid, oxalic acid and stearic acid,ethylenediaminetetraacetic acid (=EDTA),N-(2-hydroxyethyl)ethylenediaminetriacetic acid (=HEDTA),ethylenediamine-N,N′-diacetic acid (=EDDA) and nitrilotriacetic acid(=NTA) and sulfate, nitrate, nitrite and phosphate salts. The matrixused in all experiments is 2,5-dihydroxybenzoic acid.

Disadvantageously, both methods are only suitable for measuring puresubstances. This problem is addressed by Duncan et al. in theirdiscussion, where they suggest purifying the samples to be measured inorder to overcome this difficulty.

Overall however, MALDI-TOF MS is an interesting, simple and quick methodwhich gives specific information about the analyzed substances so thatit would be desirable to use MALDI-TOF MS for measuring enzymaticreactions with low molecular weight substances. Its use in highthroughput screening would be especially desirable.

It is an object of the present invention to develop a process foranalyzing enzyme-catalyzed reactions by using MALDI-TOF massspectrometry.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. PEA determination against internal standards. FIG. 1 aillustrates PEA concentration with phenylmethylamine as internalstandard. FIG. 1 b PEA concentration with illustrates d₅-PEA as internalstandard.

FIG. 2 illustrates the quantitative determination of PEA against d₅-PEAas internal standard as the ratio of analyte to internal standard.

FIG. 3 depicts the measurement of PEA against d₅-PEA as internalstandard in a relative concentration range (analyte/internal standard(A/IS)) from 0.1 fold to 2 fold.

FIG. 4 depicts a calibration line using an automated program.

FIG. 5 depicts the profile of the analyte/internal standard (A/IS)distribution across a sample on the nickel vapor-coated glass target.FIG. 5 a shows the manual application and FIG. 5 b the automaticapplication.

FIG. 6 depicts the profile of the A/IS distribution across a sample onthepolished metal target. FIG. 6 a shows the manual application and FIG.6 b the automatic application.

FIG. 7 depicts the quantitative analysis of PEA against d₅-PEA asinternal standard on different target. FIG. 7 a shows the polishedtarget, FIG. 7 b the glass target, and FIG. 7 c the plate withhydrophilic holes.

FIG. 8 shows a MALDI spectrum of a mixture of unlabeled and labeledderivatized methoxycyclohexanol.

FIG. 9 depicts the results of quantitative MALDI of MCS against d₃-MCSas internal standard.

FIG. 10 depicts the amount of methoxycyclohexanol before and afterenzymatic conversion.

FIG. 11 depicts the conversion of enantiomerically puremethoxycyclohexanol, measured at different matrix/analyte ratios.

FIG. 12 depicts the time course of the enzymatic conversion of racemicmethoxycyclohexanol using immobilized lipase.

DETAILED DESCRIPTION OF THE INVENTION

We have found that this object is achieved by a process for analyzingenzyme-catalyzed conversions of nonpolymeric substrates to nonpolymericproducts, which comprises analyzing the substrate and product of theenzyme-catalyzed conversion with the aid of MALDI-TOF mass spectrometry,with said process including the following steps:

-   a) enzyme-catalyzed conversion of a nonpolymeric substrate to a    nonpolymeric product,-   b) analysis of the substrate or product or of the substrate and    product during or after the enzyme-catalyzed conversion (a) using    MALDI-TOF mass spectrometry.

Enzyme-catalyzed conversions mean enzymatic reactions involving wholecells which may be of plant, animal, bacterial or fungal origin; yeastcells are also suitable. The enzymatic conversion may be carried out byquiescent, growing, permeabilized or immobilized cells ormicroorganisms. Enzymes are also suitable for the enzyme-catalyzedconversion. These enzymes may still be included in the permeabilizedcells or microorganisms or else be present in crude extracts. For arelatively quick and usually also relatively by-product-free conversionit is possible to use partly purified or purified enzymes, which may beused in free or immobilized form in the conversion. Preferably thereaction is carried out using free, partly purified, purified orimmobilized enzymes.

Advantageously, the process of the invention uses enzymes of enzymeclasses 1 to 6 (International Union of Biochemistry and MolecularBiology=IUB), preferred are enzyme classes 1 to 4, particularlypreferred is enzyme class 3 such as subclasses 3.1 (acting on esterbonds), 3.2 (glycosidases), 3.3 (acting on ether bonds), 3.7 (acting oncarbon-carbon bonds) and 3.11 (acting on carbon-phosphorus bonds), veryparticularly preferred are enzymes such as lipases, esterases orphosphatases such as phytases. Further advantageous enzymes can be foundin enzyme class 6.

Nonpolymeric substrates and nonpolymeric products in the process of theinvention are compounds which, in particular, are not peptides,proteins, oligonucleotides or polynucleotides or oligosaccharides orpolysaccharides or artificial or natural polymers. These nonpolymericsubstrates or nonpolymeric products possess a molar mass of less than1000 Da (=dalton), preferably less than 800 Da, very particularlypreferably less than 600 Da.

Besides the analysis of substrate and product of the reaction it ispossible and advantageous to follow enzyme reactions with the aid of theprocess of the invention, i.e. kinetic studies of enzymes can beperformed. It is further possible to determine K_(m), V_(max), enzymeselectivity, reaction yield and the effect of inhibitors on an enzymereaction. Likewise it is possible to study possible reaction parameterssuch as temperature or pH with respect to the enzyme-catalyzed reaction.

It is not necessary to purify the reaction solutions of the process ofthe invention prior to analysis by MALDI-TOF mass spectrometry. Thereaction can be measured directly. This is also true for complex samplemixtures. Likewise, it is not necessary to use pure substances for thereaction, although this is certainly possible.

It is advantageous and possible to derivatize prior to the analysissubstrates and/or products which are only poorly or not at alldetectable in MALDI-TOF MS (see Examples) and to finally analyze them inthis form. The derivatization may be carried out before or after theenzymatic reaction. Derivatization is particularly advantageous in thosecases where hydrophilic groups, which advantageously carry an additionalionizable function, are introduced into hydrophobic or volatilecompounds such as, for example, esters, amides, lactones, aldehydes,ketones, alcohols, etc. Examples of such derivatizations are conversionsof aldehydes or ketones to oximes, hydrazones or derivatives thereof orof alcohols to esters, for example with symmetrical or mixed anhydrides.This significantly extends the detection spectrum of the process.Derivatization after the enzymatic conversion makes it possible todirectly measure the original substrate of the enzymatic reaction. Byusing MALDI-TOF MS it is thus possible to analyze even substancescontaining no chromophore. Compared with other processes, this is asubstantial advantage since conventional detection processes, forexample, usually have to use artificial substrates, which contain e.g. achromophore, for visual detection, for example. When optimized for thisreaction, said substrates will probably not improve the desired naturalenzymatic conversion since optimizing this artificial reaction does notreflect the natural conditions.

In the process of the invention for analyzing enzyme-catalyzed reactionsan internal standard is advantageously added. This internal standardmakes it advantageously possible to quantify low molecular weightcompounds in the reaction solution. This standard may be added to theenzyme-catalyzed conversion before, during or after the enzymaticreaction. In this way, substrate and product or, where appropriate,other intermediates of the reaction may be analyzed and, in the end,quantified. In the end, the intermediates can also be seen as productsof the substrate employed at the beginning of the reaction. Using theprocess of the invention, it is therefore also possible to follow oranalyze enzyme reactions which catalyze successive reactions. These maybe catalyzed by one enzyme or a plurality of enzymes. By-products mayalso be analyzed.

The internal standards used are advantageously labeled substances, butchemical compounds similar to the substrates and/or products are inprinciple also suitable as internal standards. Such similar chemicalcompounds are, for example, compounds of a homologous series whosemembers only differ by, for example, an additional methylene group. Itis preferred to use substrate or product, which is labeled by at leastone isotope selected from the group comprising ²H, 13C, ¹⁵N, ¹⁷O, ¹⁸O,³³S, ³⁴S, ³⁶S, ³⁵Cl, ³⁷C, ²⁹Si, ³⁰Si, ⁷⁴Se or mixtures thereof, oranother labeled chemical compound as internal standard. Advantageously,the substrate is labeled by at least one isotope selected from the groupcomprising ²H, 13C, ¹⁵N, 170, ¹⁸O, ³³S, ³⁴S, ³⁶S, ³⁵Cl, ³⁷Cl, ²⁹Si,³⁰Si, ⁷⁴Se or mixtures thereof. For expense and availability reasons theuse of ²H or 13C as isotope is preferred. It is not necessary for theseinternal standards to be completely, i.e. fully labeled for theanalysis. Partial labeling is entirely sufficient. It is advantageousand sufficient to have labels at a distance of from 3 or more dalton to10 or less dalton. However, measurement is in principle also possible atbelow 3 dalton or above 10 dalton, but short distances may possibly leadto overlapping with the isotopes of the analyte and longer distances maypossibly lead to isotope effects. This makes measurements more difficultbut not impossible. It is advantageous, even in the case of a labeledinternal standard, to select a substance which has maximum homology,i.e. structural similarity, with the chemical compound to be measured.The greater the structural similarity, the better are the measurementresults and the more accurately ca the compound be quantified.

For the process of the invention and particularly for quantifying thesubstrates, products, intermediates or by-products present in thereaction, it is advantageous to use the internal standard in a favorableratio to the substrate, product, intermediate or by-product to bemeasured. Ratios between analyte (=compound to be measured) and internalstandard of greater than 1:15 do not improve the measurement results,they are, however, possible in principle. Advantageously, the ratiobetween analyte and internal standard is adjusted in a range of 0.1 to15, preferably in a range of 0.5 to 10, particularly preferably in arange of 1 to 5.

It is advantageous to concentrate the analytical samples on a minimumspace or on a minimum diameter in order to achieve further improvementin data point resolution and/or measurement accuracy.

The reaction samples in the process of the invention may be preparedeither manually or, advantageously, automatically by conventionallaboratory robots. Analysis by MALDI-TOF MS may likewise be carried outmanually or, advantageously, automatically. Automation of the process ofthe invention makes it possible and advantageous to use MALDI massspectrometry for the fast screening of enzyme-catalyzed reactions inhigh throughput screening. MALDI-TOF MS stands out here due to highsensitivity combined with minimum sample consumption. This method makesit possible to quickly find novel enzyme activities and novel mutants ofknown enzymes after mutagenesis, for example after conventionalmutagenesis using chemical agents such as NTG, radiation such as UV orX-ray radiation, or after site-directed mutagenesis, PCR mutagenesis orgene shuffling.

It is advantageous to use for the process of the invention carriermaterials having a value or number of roughness R_(z) of greater than 1,preferably greater than 2, particularly preferably greater than 3 andvery particularly preferably greater than 4. R_(z) is the averagedpeak-to-valley height (μm) which is the arithmetic mean value of theindividual peak-to-valley heights of 5 neighboring individually measuredsections. The peak-to-valley height is determined according to DIN 4768.These carrier materials are polished, coated or vapor-coated carriermaterials or polished and coated carrier materials or polished andvapor-coated carrier materials. The carriers comprise a materialselected from the group comprising glass, ceramics, quartz, metal,stone, plastics, rubber, silicon, germanium or porcelain. The materialpreferably comprises metal or glass.

To determine other analytical data, it is possible in the process of theinvention to additionally carry out the analysis with the aid ofmetastable fragmentation after ionization or of collision-inducedfragmentation. This makes it possible to obtain further mass data whichmake it easier or possible to identify the substrates, products,by-products or intermediates present.

It is advantageous in the process of the invention to measure thedynamics of the labeling pattern and the substrate and productconcentrations. This makes it possible to analyze the kinetics ofenzymes. In this way it is possible to determine Km and V_(max) of anenzyme.

The following examples illustrate the invention in more detail:

EXAMPLES Example 1 Lipase-Catalyzed Conversion of Rac. Phenylethylamine(=PEA) to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (=MET)

Unless described otherwise in individual examples, the experiments werecarried out as follows:

-   Matrix/analyte ratio=50 (mg/mg)-   Solvent: 50% EtOH/50% H₂O/1% HOAc/0.1% TFA (v:v:v:v)-   Internal standard (IS):d₅-PEA-   V_(total)=1 ml-   Stock solutions:    -   DHB: 140 mg/ml    -   PEA: 35 mg/ml    -   d₅-PEA: 35 mg/ml-   Pipetting schedule:

d₅-PEA rel. PEA V_(PEA) (IS) V_(d5-PEA) DHB V_(DHB) Solvent conc.[mg/ml] [μl] [mg/ml] [μl] [mg/ml] [μl] [μl] 0.1 0.014 0.4 0.14 4 7.7 55940.6 0.5 0.07 2 0.14 4 10.5 75 919 1 0.14 4 0.14 4 14 100 892 1.5 0.216 0.14 4 17.5 125 865 2 0.28 8 0.14 4 21 150 838 2.5 0.35 10 0.14 4 24.5175 811 3 0.42 12 0.14 4 28 200 784 3.5 0.49 14 0.14 4 31.5 225 757 50.7 20 0.14 4 42 300 676 7.5 1.05 30 0.14 4 59.5 425 541 10 1.4 40 0.144 77 550 406

-   -   Sample application by nanoplotter    -   Measurement: manually, 13 positions with 25 shots each

The experimental results are depicted in FIG. 2.

PEA was determined quantitatively in all experiments. It was shown thatit is possible to determine MET quantitatively when the internalstandard used is PEA; however, in this case the errors were distinctlylarger due to the different molecular structures of the two compoundsand the different ionization and flight properties associated with saidstructures. A similar behavior was observed when determining PEA againstphenylmethylamine as internal standard (FIG. 1 a). The best results wereobtained with the internal standard having maximum molecular homology,that is structural similarity, to the analyte, as for example d₅-PEA toPEA (FIG. 1 b).

Example 2 Effect of the Ratio of Analyte to Internal Standard onQuantification

a) Measurement Over a Relatively Wide Range of Relative Concentrations(0.1 to 10 Fold)

In this experiment the ratio of analyte to standard was varied from 0.1times to 10 times. The result is shown in FIG. 2. The samples wereapplied by means of nanoplotter and measured by manually approaching thespots in the MALDI. For each spot 13 positions were measured with 25shots each and then the results were added up. All concentrations weredetermined 4 times in each case. The absolute concentration of theinternal standard was 0.14 mg/ml.

-   -   Samples are prepared according to the abovementioned pipetting        schedule (Example 1) and transferred into a 96-well plate.    -   The nanoplotter is programmed such that for each concentration        50 μl of the sample solution are drawn in; four spots for each        concentration are then spotted onto different zones of the MALDI        target (quadruple determination).    -   The exact volume of each spot was not determined, but it is in        the order of 0.5 nl.    -   In order to ensure a reproducible drop formation in the        nanoplotter, the parameters for the piezocrystal were varied        with the aid of the stroboscope on the nanoplotter and        subsequent examination of the spots through a binocular        microscope. Typical parameters used in the PEA experiment were:        -   f=150 Hz (pumping frequency)        -   T=20 μs (pulse width)        -   U=60 V (amplitude)

In many cases it was possible to observe the formation of satellitespots which are a well-known phenomenon for this sample applicationtechnique but which had no effect on the measurement results. Optimumvalues vary greatly, depending on concentration ratios!

FIG. 2 depicts the quantitative determination of PEA against d₅-PEA asinternal standard. A saturation of the curve is clearly visible when theratio of analyte to internal standard becomes too high.

After a linear increase a distinct saturation of the curve can be seen.The reason for this must be seen in different signal intensities of thetwo signals at a large concentration difference. At a highanalyte/standard ratio (=A/IS) it is thus possible, for example, for theanalyte signal to be at the detector limit (256 counts/shot), whereasthe signal of the internal standard is just above the required qualitycriterion of the signal-to-noise ratio.

b) Measurement Over a Narrow Relative Concentration Range

In this experiment, the ratio of analyte to internal standard was variedfrom 0.1 times to 2 times. The result is shown in FIG. 3 [measurement ofPEA against d₅-PEA as internal standard in a relative concentrationrange (A/IS) of 0.1 times to 2 times]. Sample preparation andmeasurement made use of the same parameters as in the previousexperiment (Example 2a). As in Example 2a, there was in the lower rangea distinct linear dependence of the signal intensity ratio of A to IS onthe relative concentrations of the two components. This range thereforeis also advantageous for enzyme assays. Since the startingconcentrations are known in an enzyme assay, it is possible readily tocalculate the concentration of the internal standard or the ratio ofanalyte, for example product, to internal standard, in order to obtain afavorable ratio to the analyte. The relative standard deviation in thisexperiment was usually less than 5%.

Example 3 Sample Application Using a Nanoplotter

Preparing the samples by means of a nanoplotter is intended to apply theminimum amount of the matrix/analyte mixture and to achieve the quickestpossible solvent evaporation, which should reduce separation of the twocomponents.

It was possible to show that the nanoplotter permits a simple and rapidpreparation of MALDI samples which can lead to reproducible results forquantification. The matrix crystals are distinctly smaller compared withmanual preparation. In addition, analyte distribution in the matrixseems to be somewhat more homogeneous than in manual preparation (datanot shown). However, this preparation type frequently led to theformation of “fried egg-like” structures, i.e. matrix and analyte form aring which contains no (or at least distinctly less) ionizable materialin its center. The formation of such structures seems to be dependent onmatrix/analyte concentrations and the solvent used. It was not possiblein this connection to establish general rules; it can be said, however,that this phenomenon is also observable in the manual preparation.

However, it was possible to detect two differences between manualpreparation and nano-preparation. Thus, it was possible to show that formanual preparation a slightly larger range in the ratio of analyte tointernal standard leads to a linear correlation, whereas for thepreparation by means of the nanoplotter the above-described saturation(Example 2, FIG. 2) appears quite early. In contrast to this therelative standard deviation was smaller for the nano-preparation thanfor manual preparation. Both methods provide comparable and satisfactoryresults. All examples were carried out both manually and automatically.

Automated Recording of Data

For the automated recording of data the program AutoXecute™ was usedwhich is part of the control software of the Bruker Reflex III MALDI-TOFmass spectrometer, and which permits the automated recording of MALDIspectra. It was possible to optimize the parameters of this software formeasuring low molecular weight compounds. In this connection, thefollowing items, inter alia, were considered for the automatic dataacquisition: saturation effects of the signals of the matrix, theanalyte or the internal standard; saturating the detector limit; laserintensity; peak resolution; signal-to-noise ratio; baseline noise, andadding up the appropriate signals.

The following parameters were used for the automated recording of data:

-   -   Laser attenuation: 69-63    -   Recording format: large spiral    -   Number of peaks added up: 100    -   Optimum range of A/IS ratio: 1-5    -   Resolution≧1400    -   Signal-to-noise ratio>6    -   Baseline noise=200 a.i.

FIG. 4 depicts, by way of example, a calibration line which was recordedby means of said parameters. The samples were identical to thosemeasured in Example 2b (FIG. 3). Sample preparation was carried out bymeans of the nanoplotter; the recording spiral had not been optimizedfor such small sample drops, and therefore a large number of laser shotsmissed the samples (for each concentration four spots were measured).Nevertheless, a result was obtained which was analogous to the classicalrecording technique depicted in FIG. 3; in this classical recordingtechnique the measured spots were approached or sighted manually andshot at 13 different positions by 25 laser shots each. The signals ofthese 13×25 measurements were added up by the MALDI spectrometer andrepresent the result of a single measurement. Manual sample preparationgave analogous results. Thus the use of the program AutoXecute™ permitsthe automated quantification of low molecular weight compounds.

Example 4 Influence of the Target Characteristics

In order to study possible effects of the target characteristics onsample homogeneity, four different MALDI targets were employed:

-   -   Unpolished metal target    -   Polished metal target    -   Nickel vapor-coated glass target    -   Hydrophobically coated plate with small holes (the holes for        their part have the same characteristics as the unpolished        target.)

The experiments were carried out as described in Examples 1 and 2. Theunpolished target proved to be unsuitable in both manual preparation andpreparation by means of the nanoplotter, since the grooves on thesurface caused an inhomogeneous crystallization.

When using the nanoplotter, it was impossible to detect any differencesbetween polished metal target and glass target regarding samplehomogeneity and analyte distribution in the matrix, so that in this caseusing either plate leads to equivalent results in quantification.However, the glass target had the advantage that very small sample spotswere more clearly visible in the video microscope of the MALDIapparatus.

Results:

One profile of the analyte/IS ratio across a manually applied spot wasplotted for each of the different concentrations (FIGS. 5 a+5 b, 6 a+6b). FIG. 5 depicts the profile of the analyte/internal standard (=A/IS)distribution across a sample on the nickel vapor-coated glass target.FIG. 5 a shows the manual application and FIG. 5 b the automaticapplication.

Comparison of FIGS. 5 a+5 b with FIGS. 6 a+6 b shows that in the case ofmanual preparation the distribution of analyte and standard on the glasstarget is not as homogeneous as on the polished target.

It can be seen that the intensity distribution at low analyte/matrixconcentrations is uneven and that the highest signal intensity isvisible at the edge of the spots (“fried-egg shape”). At higherconcentrations, sufficiently strong signals are also detected in thecenter of the spot. This behavior is analogously seen in the preparationusing the nanoplotter.

FIGS. 7 a) to c) indicate that the calibration curves are comparableindependently of target composition. FIGS. 7 a)-c) show the quantitativeanalysis of PEA (7μg/ml to 1400 μg/ml) against d₅-PEA (140 μg/ml) asinternal standard on different targets. The samples were appliedmanually (0.34 μl per well). The average standard deviation was about10% for all three targets. The best data (relative standarddeviation≦5%) were obtained, as already described above, for A/IS ratiosfrom 1 to 5. Very good results were obtainable when using the targethaving small hydrophilic holes (R²=0.9994). Concentrating the sample ona smaller area thus achieves a gain in accuracy.

Example 5 Lipase-Catalyzed Preparation of Enantiomerically Pure1S,2S-methoxycyclohexanol (MC)

The following reaction which is depicted in diagram II and catalyzed bya lipase was used as a model reaction for a MALDI-TOF MS-based methodfor quantitative screening of enzymatic reactions.

To establish a screening assay using the reaction depicted in diagramII, it was firstly determined whether it is possible at all to detectthe molecules involved in the reaction by means of MALDI MS. Inaddition, a number of different matrices were tested which for theirpart have acidic or basic properties and should facilitate or improvedetection. The compounds studied of the reaction were:

-   -   Vinyl decanoate (L)    -   Methoxycyclohexanol (MC)    -   Methoxycyclohexanyl decanoate (MCL)

Additionally, it was attempted in one case to induce the formation ofsodium adducts by adding NaCl. In the case of two matrices, SDS wasadditionally added.

In all cases the attempt was made to measure in both positive andnegative mode.

Table 1 lists the molar masses and the expected ions (calculated) of theindividual compounds.

TABLE 1 Theoretically expected signals Mono- isotopic Molecularmolecular Compound formula weight. [M + H]⁺ [M + Na]⁺ [M − H]⁻ VinylC₁₂H₂₂O₂ 198.162 199.169 221.152 197.154 decanoate (L) Methoxy- C₇H₁₄O₂130.099 131.107 153.089 129.091 cyclohexanol (MC) Methoxy- C₁₇H₃₂O₃284.235 285.243 307.225 283.227 cyclohexanyl decanoate (MCL)

Table 2 lists the different matrices used for carrying out themeasurements and the results of these measurements.

TABLE 2 Matrices used and results of measurements in positive andnegative modes. +: Signal detected, −: signal not detected, *overlapping of a (theoretical) signal with a matrix signal. L L MC MCMCL MCL Compound (pos) (neg) (pos) (neg) (pos) (neg) 2,5-DHB — — — — — —2,5-DHB + SDS — — — — — — SA — — — — — — CCA — — — — — — CCA, (M/A) = 1— — — — — — CCA + SDS — — — — — — CCA + NaCl — — — — — —2-amino-5-nitropyridine — — — — — — Dithranol/AgTFA — — — — — —Abbreviations in Table 2: 2,5-DHB = 2,5-Dihydroxybenzoic acid SDS =sodium dodecyl sulfate SA = sinapinic acid CCA =α-cyano-4-hydroxycinnamic acidConditions of Measurement:

-   -   Measurement in positive and negative mode    -   All matrix solutions were made up freshly:        -   CCA, SA, DHB, dithranol/AgTFA: in 90% acetonitrile/10%            water/0.1% TFA        -   2-amino-5-nitropyridine: in 90% acetonitrile/10% water    -   The matrix-to-analyte ratio (M/A) was varied:        -   M/A=100 (mg/mg)        -   M/A=30 (mg/mg)        -   M/A=1 (mg/mg)    -   Stock solutions of the analytes (in acetonitrile) were prepared.        Evaluation of Results:

Despite varying the conditions (matrices, solvent, pH) it was impossibleto detect analyte signals either in positive or negative mode. Apossible reason for this is possibly the volatility of the analytesunder the MS conditions (high vacuum). Another possible explanation maybe the apolar nature of the compounds which makes accessibility of saidcompounds to MALDI analysis very difficult. In order to be able tofollow the reaction, the methoxycyclohexanol was therefore derivatized.For measurement in MALDI MS, methoxycyclohexanol (=MC) was derivatizedto give the corresponding methoxycyclohexanyl sulfobenzoate (MCS).

Derivatization of methoxycyclohexanol had two main purposes. Firstly,the derivative should become less volatile and secondly, an ionizablegroup should be introduced into the molecule. Preliminary experimentsusing various aromatic acid chlorides (benzoyl chloride, p-nitrobenzoylchloride) gave unsatisfactory results. The reaction of the alcoholicfunction with a mixed anhydride (2-sulfobenzoic anhydride (SBA, Bagreeet al., FEBS Lett., 120, 1980: 275-277) finally provided the desiredsuccess. Diagram III depicts a diagrammatic representation of thederivatization.

The stereochemistry of methoxycyclohexanol was not taken into account.The model reaction used the following enantiomerically pure compound:

Experimental Procedure

-   -   Methoxycyclohexanol (MC)=130.1 g/mol    -   2-Sulfobenzoic anhydride=184.17 g/mol    -   d₃-MC=134.1 g/mol (calculated as d₄, since the hydroxyl function        is likewise deuterated)

-   1. Initially charging 0.5 g (3.84 mmol) of methoxycyclohexanol (MC)

-   2. Dissolving 777.9 mg (1.1 eq., 4,224 mmol) 2-sulfobenzoic    anhydride in 0.5 ml acetonitrile (is dissolved completely during the    course of the reaction)

-   3. Adding solution 2) to solution 1)

-   4. Shaking at room temperature for 2 hours

-   5. When sample starts crystallizing: dissolving in 2 ml of    acetonitrile/1 ml of water (very readily soluble)

-   6. Preparing MALDI sample

Description for Preparing the Standard (d₃-MCS): Analogously Weight(d₃-MC): 515 mg

MALDI Sample Preparation:

a) Theoretically Expected Masses of the Derivative

M_(monoisotopic): 314,08

-   -   [M+H]+: 315,09    -   [M+Na]+: 337,07    -   [M−H]−: 313,07        b) Search for Suitable Matrix

Various matrices were tested which have either acidic or basicproperties. Measurements were carried out in negative mode.

TABLE 3 Matrices for measuring the derivatized methoxycyclohexanol (MCS)Peaks in Matrix pH measured range evaluation 2, 5-DHB acidic yesunsuitable SA acidic yes unsuitable CCA acidic yes unsuitable HABAacidic yes unsuitable Trihydroxyacetophenone acidic yes unsuitable2-Amino-5-nitropyridine basic yes unsuitable ATT (6-aza-2-thiothymine)basic 314, 91 suitable

The analyte was found in all matrices; the best results and minimuminterference from matrix signals, however, were clearly obtained whenusing ATT.

c) Obtained When Spotting Using the Nanoplotter

The samples were spotted onto the MALDI target by means of a nanoplotterusing the conditions of the DHB/PEA solutions used earlier (seepipetting schedule and nanoplotter description, Example 1). Sometimessatellite peaks were detected which had, however, no negative effect onthe measurement. The peaks were homogeneous (visual impression fromMALDI microscope and binocular).

In the latter measurement no serious variations in the A/IS ratio withinthe spots were detected (except for the usual variation).

d) Sample Preparation for Quantitative MALDI

Since the true analyte concentration after the reaction (=yield orconversion) was unknown, the various solutions were mixed with eachother in different volume ratios.

The matrix solution used was a saturated solution of ATT in AcCN/H₂O(1:1, v:v). A calculation of the accurate matrix/analyte ratio was notpossible in this way, but said ratio was kept constant over the entirerange measured.

MALDI Measurement:

a) Conditions of Measurement:

-   -   negative mode    -   reflector mode    -   a polished target was used        b) MALDI Spectrum of the Derivatized Methoxycyclohexanol (MCS)

FIG. 8 shows a MALDI spectrum of a mixture of unlabeled (MCS,m/z=331.01) and labeled (d₃-MCS, m/z=316.02) derivatizedmethoxycyclohexanol (matrix: ATT).

Fragments or adducts were not visible under the chosen conditions ofmeasurement. Only the monoisotopic peak was used for subsequentquantitative evaluation, the isotope peaks were neglected.

c) Quantitative Measurement

12 positions per spot were shot at, 25 shots being added up in eachcase; the sum of 12*25 shots was then evaluated. Four spots were shot atfor each concentration. Two outliers were found (at rel. conc. 0.2 andrel. conc. 1.4) which were not taken into account in FIG. 9.

FIG. 9 depicts the results of quantitative MALDI of MCS against d₃-MCSas internal standard.

A linear correlation between the ratio of analyte signal intensity tointernal standard intensity and the concentrations of the two compoundsrelative to each other was found. The derivatizing reaction and theMALDI parameters used (matrix etc.) permitted a quantitative evaluationof the reaction. This was confirmed using a commercially availablelipase (Boehringer, Mannheim, Germany).

Procedure:

-   -   1.01 mmol (200 mg) vinyl decanoate and 1.01 mmol        methoxycyclohexanol (1.01 mmol) were mixed.    -   Enantiomerically-pure MC was used.    -   Sample split        -   a) control        -   b) enzyme reaction    -   Addition to b) of 50 mg of enzyme. Enzyme: Chirazym L-2, c.-f.,        C2, Lyo, Boehringer Mannheim; lipase from Candida antarctica,        fraction B, approx. 4.5 kU/g carrier;    -   Addition of 250 μl of hexane to each    -   Shaking at room temperature for 24 h    -   Solutions were filtered with suction, and subsequently washed        with 250 μl of AcCN each    -   Addition of 0.5 mmol of d₃-methoxycyclohexanol (67 mg,        calculated as d₄-MC) each    -   Addition of 1.5 mmol of 2-sulfobenzoic anhydride (SBA, 276.6 mg)        each in 500 μl of AcCN    -   Stirring for 20 h    -   (Red coloring of the mixtures detected)    -   Addition of 27 μl of water (=1.5 mmol) each to stop        derivatization    -   Mixing of MALDI sample, matrix: saturated ATT, AcCN/water, 1:1        -   a) 20 μl of sample+50 μl of matrix+150 μl of AcCN (M/A: low)        -   b) 20 μl of sample+100 μl of matrix+100 μl of AcCN (M/A:            medium)        -   c) 20 μl of sample+200 μl of matrix (M/A: high)    -   Applying sample onto target: nanoplotter, polished target    -   MALDI measurement, conditions as for model reaction        Results:

A distinct reduction in the amount of methoxycyclohexanol after theenzymatic reaction compared with the control reaction was detectable(FIG. 10). It was also possible to demonstrate that the M/A ratio had nogreat influence on this result. At low M/A ratio the spectra had merelya distinctly lower quality (distinctly poorer signal-to-noise ratio),which can also explain the small deviation from the measurements atmedium and high M/A ratios (FIG. 11). FIG. 11 depicts the conversion (inpercent) of enantiomerically pure methoxycyclohexanol, measured atdifferent matrix/analyte ratios. The deviation at low M/A ratio isprobably due to the poor signal-to-noise ratio for this measurementseries.

Example 6 Kinetics of a Lipase-Catalyzed Reaction in Microtiter Plates:Immobilized BASF Lipase

The above-specified racemate separation of methoxycyclohexanol wascarried out in this experiment in a microtiter plate to which a lipasefrom Burkholderia plantarii had been noncovalently immobilized (BASF,DSMZ 8246).

Experimental Procedure:

-   -   Mixing of 2 g of methoxycyclohexanol and 2.26 g of vinyl        decanoate (V_(total)=4.275 ml); 200 μl of this solution        correspond to 93.5 mg of MC    -   200 μl of this mixture were pipetted into each well of the        microtiter plate, the plate was sealed using plate sealer,        covered and incubated at room temperature (=28° C.) and 150 rpm        on an orbital rotation shaker    -   At time t 80 μl were taken from a well (this corresponds to 37.4        mg=2.875*10⁻⁴ mol of MC). (Furthermore, at the same time 100 μl        were transferred into a GC sample vial, overlaid with ethyl        acetate (1 ml) and stored at −20° C. until GC analysis).    -   To 80 ml of sample 38.6 mg of d₃-MC were pipetted (=2.875*10⁻⁴        mol)    -   These samples were stored intermediately at −20° C. until        completion of the enzymatic reaction so that all derivatization        mixtures started at the same time.    -   179 mg of SBA in 400 ml of acetonitrile were pipetted into each        of the mixtures which were then shaken at room temperature        overnight.    -   20 μl of the derivatization solution were then mixed with 200 μl        of saturated ATT solution (water/AcCN, 1:1) in each case and        spotted onto the polished metal target by means of the        nanoplotter.        Results:

A distinct reduction in the total amount of methoxycyclohexanol wasdetectable during the course of the enzymatic reaction using immobilizedlipase (FIG. 12).

The measurement determined the total amount of MC which was stillpresent after the reaction and which can be both racemic substrate andenantiomerically pure product. The method of the invention made itpossible to measure a reduction in the total amount ofmethoxycyclohexanol by 18% after a reaction time of 30 h.

The results from all experiments make it possible to derive thefollowing:

-   -   Signals should advantageously be recorded at a signal-to-noise        ratio of greater than 3; as a quality indicator this ratio        advantageously should be greater than 10, which is achievable        without problems under the conditions studied.    -   Laser attenuation should advantageously be set to a minimum        (above threshold), in order to prevent detector saturation and        excessive fragmentation of the analyte.    -   It is often advantageous to adjust the change in laser        attenuation during a series of measurements, in order to prevent        saturation of the signal intensities (e.g. rel. conc=0.1, attn:        60/61; rel. conc.=10, attn.: 65/66). This option is also        available in the Bruker AutoXecute™ program.    -   Widening the laser reduces the number of firing positions needed        for each spot; it is, however, impossible to cover a drop by one        laser position.    -   Sample homogeneity was greater using the nanoplotter compared        with using manual preparation.    -   The relative errors are below 5% when using the nanoplotter.        Optimum results for the lipase reaction are within a narrow        concentration range. This means that the analyte concentration        ought to be advantageously between 0.1 times and double the        concentration of the internal standard.    -   Due to grooves on the surface, unpolished metal targets are        distinctly inferior to polished targets with respect to        homogeneous sample preparation and thus unsuitable for        quantitative measurement. They are however suitable for        qualitative measurement.

Unless stated otherwise in the examples, the experiments were carriedout using the following equipment and chemicals:

MALDI Mass Spectrometer:

-   -   Bruker Reflex III MALDI-TOF, Bruker, Bremen, Germany    -   N₂ laser, λ=337 nm    -   Scout 384-well target    -   optionally:        -   polished Bruker standard metal target or        -   Bruker glass target (prototype) or        -   Bruker standard metal target            Nanoplotter:    -   GeSim micropipetting system nanoplotter, type: P30-x-D    -   Gesellschaft für Silizium-Mikrosysteme mbh,        Groβerkmannsdorf/Rossendorf, germany    -   piezoelectric micropipette from the same company        Chemicals:    -   2,5-DHB: Aldrich    -   ATT: Aldrich    -   2-sulfobenzoic anhydride: Fluka    -   all other chemicals: BASF (unless stated otherwise)

1. A process for analyzing enzyme-catalyzed conversions of nonpolymericsubstrates to nonpolymeric products, which comprises analyzing asubstrate or a product or a substrate and product of theenzyme-catalyzed conversion with the aid of Matrix-Assisted LaserDesorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry, withsaid process comprising the following steps: a) enzymatically convertinga nonpolymeric substrate to a nonpolymeric product; and b) analyzing thesubstrate or product or the substrate and product during or after theenzyme-catalyzed conversion of step (a) using MALDI-TOF massspectrometry, wherein the analysis is carried out on a polished orcoated carrier material having a roughness of Rz>I and wherein thesubstrate or product or the substrate and product are mixed with aninternal standard, wherein nonpolymeric substrates and nonpolymericproducts are not peptides, proteins, oligonucleotides, polynucleotides,oligosacoharides, polysacoharides, natural polymers or artificialpolymers.
 2. The process as claimed in claim 1, which comprises addingthe internal standard before the start of the enzyme-catalyzedconversion or during or after conclusion of the enzyme-catalyzedconversion and analyzing the substrate or product or substrate andproduct in the presence of this internal standard.
 3. The process asclaimed in claim 1, which comprises analyzing substrates or productshaving a molar mass of <1000 dalton.
 4. The process as claimed in claim1, which comprises quantifying the substrate or product or the substrateand product of the enzyme-catalyzed reaction.
 5. The process as claimedin claim 1, which comprises using free or immobilized enzymes, crudeextracts or whole cells for the enzyme-catalyzed reaction.
 6. Theprocess as claimed in claim 1, wherein the analysis is carried out on apolished and coated carrier material.
 7. The process as claimed in claim1, wherein the carrier material comprises a material selected from thegroup consisting of glass, quartz, metal, stone, rubber, silicon,germanium and porcelain.
 8. The process as claimed in claim 1, whichcomprises derivatizing the product prior to the analysis.
 9. The processas claimed in claim 1, wherein the process is carried out manually orautomatically.
 10. The process as claimed in claim 1, which comprisesusing the process in high throughput screening.
 11. The process asclaimed in claim 1, which comprises using substrate or product, which islabeled by at least one isotope selected from the group consisting of²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁴S, ³⁶S, ³⁵Cl, ³⁷Cl, ²⁹Si, ³⁰Si, and ⁷⁴Se ormixtures thereof, or another labeled chemical compound as internalstandard.
 12. The process as claimed in claim 1, which comprises usingsubstrate labeled by at least one isotope selected from the groupconsisting of ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, ³³S, ³⁴S, ³⁶S, ³⁵Cl, ³⁷Cl, ²⁹Si,³⁰Si, and ⁷⁴Se, or mixtures thereof as substrate.
 13. The process asclaimed in claim 1, wherein the analysis is additionally carried outwith the aid of metastable fragmentation after ionization or ofcollision-induced fragmentation.
 14. The process as claimed in claim 1,wherein the coated carrier material is vapor-coated.
 15. The process asclaimed in claim 6, wherein the coated carrier material is vapor-coated.